Alteration of taste perception, food neophobia and oral microbiota composition in children with food allergy

Currently, the mechanisms underlying sensory perception and sensory performance in children with food allergies are far from being understood. As well, only recently, single research afforded the oral host-commensal milieu, addressing oral microbial communities in children with peanut allergies. To bridge the current gaps in knowledge both in the sensory and microbial fields, a psychophysiological case–control study was performed in allergic children (n = 29) and a healthy sex-age-matched control group (n = 30). Taste perception, food neophobia, and liking were compared in allergic and non-allergic children. The same subjects were characterized for their oral microbiota composition by addressing saliva to assess whether specific profiles were associated with the loss of oral tolerance in children with food allergies. Our study evidenced an impaired ability to correctly identify taste qualities in the allergic group compared to controls. These results were also consistent with anatomical data related to the fungiform papillae on the tongue, which are lower in number in the allergic group. Furthermore, distinct oral microbial profiles were associated with allergic disease, with significant down-representations of the phylum Firmicutes and of the genera Veillonella spp., Streptococcus spp., Prevotella spp., and Neisseria spp. For the first time, this study emphasizes the link between sensory perception and food allergy, which is a novel and whole-organism view of this pathology. Our data indicated that an impaired taste perception, as regards both functionality and physiologically, was associated with food allergy, which marginally influences the food neophobia attitude. It is also accompanied by compositional shifts in oral microbiota, which is, in turn, another actor of this complex interplay and is deeply interconnected with mucosal immunity. This multidisciplinary research will likely open exciting new approaches to therapeutic interventions.

Taste sensitivity. To explore whether allergic conditions dealt differently with gustatory abilities, differences in both single taste scores (Sweet Taste Score, Sour Taste Score, Salty Taste Score, and Bitter Taste Score) and Total Taste Score (TTS) between allergic and controls separate Mann-Whitney U tests were performed. Raw scores, distributions, and significant comparisons between allergic and controls for both TTS, single taste scores, and Fungiform Papillae (FP) counts are displayed in Fig. 1.
Overall, the main effect of allergic condition on single taste scores was observed, with allergic subjects showing a lower recognition ability for Sour Taste Score (Mann-Whitney U value = 168, p < 0.001), Salty Taste Score (Mann-Whitney U value = 282, p < 0.01) and Bitter Taste Score (Mann-Whitney U value = 238, p < 0.01). These difficulties of being allergic in correctly identifying the different taste qualities resulted in considerably worse global taste performances (TTS) compared to controls (Mann-Whitney U value = 141.5, p < 0.001). The main effect of allergic conditions on FP count was also detected (t = 8.91, p < 0.001), with controls showing a greater FP density than allergic children.
Considering sex-related differences within each group, according to the Mann-Whitney test, no significant differences (p > 0.05) in gustatory abilities were found. www.nature.com/scientificreports/ Children's food neophobia, liking, and taste sensitivity. To assess whether Group-related (allergic vs controls) and sex-related differences were associated with Food Neophobia, a 2-way ANOVA was performed. Food neophobia attitudes appeared to be marginally influenced by the allergic condition (p = 0.10), showing a higher neophobic attitude for allergic than controls. No significant difference between boys and girls was found regarding the food neophobia score, nor for the interaction.
Foods characterized by sweet (mean liking = 5.2) and fatty (mean = 5.1) stimuli obtained the highest liking scores, while foods with a typical bitter (mean = 3.8) profile were the least liked. The sour (mean = 4.8) foods were scored above the foods characterized by bitterness. A double-centered PCA was used to map the children's liking scores. The first two principal components accounted for 40.5% of the variability. Two clusters of Sweet-Salty-Fatty (52.5%, n = 31) and Bitter-Sour (47.5%, n = 28) Likers (Fig. 2) were defined on the basis of PCA loading scores.
Taxonomic structure and ecological parameters of oral bacterial communities in allergic children. For investigating the composition of oral bacterial communities of allergic children (ALL), comparing it to that of sex-and age-matched controls (C), we extracted salivary DNA to produce and sequence 59 amplicons (29 from ALL patients, 30 from C), comprising the V3-V4 regions of the 16S rRNA gene. A total of 35,274 OTUs at the 97% homology level were obtained after the application of low count and low variance filters, clustered in 15 bacterial phyla, 28 classes, 57 orders, 119 families, and 257 genera. Supplementary Tables 1-5 reported the relative abundances of, respectively, phyla, classes, orders, families, and genera in individual samples.
The average relative abundance for the most represented phyla, families, and genera in ALL and C groups is shown in Fig. 3.
Concerning phyla, the two groups display comparable abundances of Bacteroidetes, Proteobacteria, and Actinobacteria. ALL patients appear characterized by the reduction of Firmicutes (25% vs 29.2% in controls) and the expansion of Candidatus Saccharibacteria (formerly known as TM7, belonging to the so-called Candidate Phyla Radiarion, CPR within the bacterial domain) and of a heterogeneous group of sequences representative of unclassified bacterial phyla. Concerning the taxonomic rank of families, the most abundant in both groups is Prevotellaceae (13.7% in C and 12.1 in ALL). Allergic children present a decrease in Neisseriaceae (class: Betaproteobacteria, 8.6% vs 10.8% in C), in the Clostridiales family of Veillonellaceae (9.2% vs 10.7 in C), and the Lactobacillales family of Streptococcaceae (4.6% vs 6% in C). On the other hand, they are characterized by the expansion of an uncharacterized bacterial family belonging to TM7 (3.8% vs 2.5%) and of unclassified bacterial families that, as for phyla, account for 9.2% of the diversity in patients (vs 5.6 in controls). Concerning genera, the picture reflects what is described above for families, with allergic patients characterized by a reduction of Prevotella (9.4% vs 11.4% in C), Veillonella (5.8% vs 7.4%), Neisseria (4.3% vs 6%), Streptococcus (4.1% vs 5.2%). On the contrary, in ALL patients, the expansion of unclassified taxa and TM7 is also seen at the genus level, with the same relative abundances observed for the higher taxonomic levels. The comparison of within-sample diversity (α-diversity) indexes evidenced a trend of increasing values in patients that reached the statistical significance for the richness index Chao1 (average for ALL: 1881,48; average for C: 1490,2; Mann-Whitney U, p = 0.00033).β-diversity was computed to determine how bacterial taxa were differentially distributed in the two groups. A clear separation (verified by the PERMANOVA test) was observed between the two groups at all taxonomic rankings(all p-values < 0.001). Figure 4 shows the Principal Coordinates Analysis (PcoA) for the phyla, families, and genera taxonomic levels.
The differential taxa abundance between allergic patients and controls was also investigated using Mann-Whitney U and Kruskal-Wallis Rank Sum non-parametric tests. Table 2 lists the taxa displaying significantly different relative abundances in ALL and C groups.
Taxa significantly depleted in ALL children comprehend the genus Streptococcus and its higher taxonomic rankings till the phylum Firmicutes; the genus Neisseria and the corresponding family (Neisseriaceae), order (Neisseriales) and class (Betaproteobacteria); the genera Prevotella and Veillonella. The most statistically significant www.nature.com/scientificreports/ difference between the two groups is represented by the enrichment, in ALL patients, of sequences not taxonomically resolved by the V3-V4 amplicon sequencing. This enrichment accompanies the ALL group at all taxonomic rankings, with the same relative abundances and an extremely significant p-value. The taxonomic identity of these sequences was further investigated by phylogenetically comparing them to the 16S sequences contained into the HOMD database (Human Oral Microbiota Database). Supplementary Fig. S1 shows the resulting phylogenetic tree, from which it emerges that these "unclassified sequences" form a monophyletic group and clusterize within Candidatus Gracilibacteria (formerly known as GNO2), another phylum within the CPR radiation. Some of these sequences were retrieved only from one of the two groups (C or ALL), while others from both, without a clear clustering.

Discussion
Multifactorial causes underlie FA, which represents one of the most crucial causes of burden to children and their families. In this study, the hypothesis that differences exist in the taste perceptions of foods and in the microbial communities dwelling in the oral cavity between allergic and non-allergic children was investigated for the first time. Food antigens and the immune system have the oral cavity as the first meeting place. Here, the functional roles of immunity and taste overlap, deciding what to 'allow' in and what to reject 34 . Thus, it is foreseeable that these systems may interact and influence each other. For example, it is known that taste cells and their signaling cascades are usually involved in auto-and hyper-immune diseases, over-expressing many genes associated with inflammation and innate immune response 35 . Moreover, in the past years, an activated inflammatory response has been correlated with impairments in taste perception in various chronic diseases, such as obesity 20,36 , diabetes 19,37 , and, more recently, the novel coronavirus infection [38][39][40] . Our results, which highlighted a reduced taste perception in children with food allergies, especially for bitter, sour, and salty tastes, support this assumption.
Taste perception was also evaluated by counting the number of fungiform papillae/cm 2 . Fungiform papillae are the gustatory anatomical structures containing taste buds and are mainly located in the anterior part of the tongue. The differential chemosensory perception among individuals has been previously linked to different fungiform papillae sizes and densities 41 . Our data supported the assumption that a direct correlation between fungiform papillae and taste perception might exist since allergic children presented a significantly lower papillae density besides impaired taste perception. This finding could be linked to the chronic low-grade inflammation reportedly associated with food allergy, which might impair the homeostasis of taste buds, decreasing their number both in the circumvallate and in the fungiform papillae, thus leading to alterations in taste. This hypothesis Taxonomic composition of the salivary microbiota in allergic (ALL) patients compared to sex-and age-matched healthy controls (C). Average relative abundance of the most represented phyla, families and genera identified in the two groups. Only taxa whose relative abundance is > 3% in at least one group are included.  www.nature.com/scientificreports/ needs further mechanistic research and a better understanding of the interactions between taste and immune systems. However, even in the absence of studies dealing directly with food allergies, indirect support comes from previous research that associated respiratory allergies and hypersensitivity to impairments in olfactory and gustatory functions 42,43 .
Although a matter of debate in the last four decades, it is still unclear whether taste sensitivity measurements relate to food liking, and studies involving children and adolescents are still limited 44,45 . As expected, based on the list of 16 food items with specific taste profiles, children liked most foods, typically sweet, fatty, and salty 44,46 . We also evidenced some links between taste sensitivity and food liking, even though only related to salty taste. Indeed, Sweet-Salty-Fatty Likers seemed less sensitive to salty, presenting more difficulties in correctly identifying it than Bitter-Sour Likers. However, research about the effect of taste sensitivity on food liking is scarce and contradictory, both in adulthood and childhood, with some studies evidencing a relationship between some taste qualities (e.g., 29,47,48 ) and others not (e.g., 44,49,50 ). Possible explanations could lie in the difficulty of studying taste sensitivity in children and that other factors besides taste sensitivity could influence taste preferences (i.e., familiarity, environmental factors, role models of parents and siblings, and previous taste exposure) 51 .
With regard to eating habits, it has been reported that food neophobia significantly determines allergic children's eating habits, influencing later in life their diet quality 14,15 . Owing to the allergy to a certain type of food, some individuals, especially children and adolescents, may continue not introducing the offending food even after having outgrown their food allergy, limiting one's food preferences. With these premises, the present study compared the degree of food neophobia between allergic children and controls using a standardized scale validated on a large group of Italian children 52 . Our findings indicated that the allergic condition appeared to marginally influence the food neophobia attitude, with a higher, although not significant, neophobic attitude for the allergic group. This difference, even if represented only by a trend in our study, has been previously reported in a larger population of children diagnosed with one or more food allergies 13 , and in adults diagnosed with celiac disease 53 . However, due to the novelty of the issue of food neophobia in a clinical context, and the presence of controversial results (e.g., 54 arrived at opposite conclusions), it will be important to further evaluate this aspect in future studies.
In addition to sensory characteristics, our analysis of the oral milieu revealed that allergic patients displayed compositional shifts in oral microbiota profiles compared to non-allergic controls. The taxonomic picture retrieved in allergic patients is characterized by a significant down-representation of the phylum Firmicutes and, Table 2. Taxa displaying significantly different relative abundances in ALL and C. Wilcoxon-Mann-Whitney and Kruskal-Wallis Rank Sum non-parametric tests were applied, using a significance threshold (p-value) set to 0.05. For each taxon, the immediately higher taxonomic ranking and the phylum are indicated. www.nature.com/scientificreports/ within it, of the genera Veillonella and Streptococcus (with all it is corresponding higher taxonomic rankings); of Prevotella spp. and of Neisseria spp. (and all the higher taxonomic rankings till the class Betaproteobacteria). Except for Neisseria (see below), our data agree with Ho and colleagues' study 32 . They analyzed the oral milieu and the salivary microbiota in a cohort of children suffering from peanut allergy, recruited at the Mount Sinai Hospital, NY 32 . The fact that the same taxonomic changes characterize the oral microbiota of two cohorts of pediatric patients with different patterns of food allergies and with other geographic origins and eating habits (among the major "modifiers" of the microbiota community structure 55 ) is noteworthy and makes it reasonable to hypothesize that such compositional shifts are strictly related to the state of food allergy itself. Ho and colleagues 32 , who also evaluated the oral metabolite and immunological profiles, linked the decreased abundances of Prevotella spp. and Veillonella spp. to reduced oral SCFA levels. Additionally, Prevotellaoral abundance in children suffering from peanut allergy negatively correlated with the oral secretion of Th2 cytokines, such as interleukin 4 (IL-4), IL-5, and IL-13, and thus associated with the skew of oral immunity towards a Th2 milieu 32 . Notably, the same genera, together with Streptococcus, appeared depleted in the gut microbiota of children with food sensitization in early life 26,56,57 . The different results on Neisseria spp., whose oral prevalence resulted in an increase in allergic children from the Ho et al. work 32 and decreased in our cohort, could be linked, in addition to the heterogeneous provenience of the populations under study, to the limited taxonomic resolution of 16S amplicon metagenomics, and the sequencing, in the two works, of different combinations of Neisseria species with different roles (e.g., as pathobionts or commensals). As commonly noticed in works based on amplicon sequencing, a gap in the detection of microbial diversity emerges from our data. Indeed, a relevant portion of the community, accounting for 5.2% of bacterial lineages in controls and almost doubled in allergic patients (9.2%), consistently from phyla to genera, is represented by "unclassified bacteria". This observation could explain the data on α-diversity which, contrary to expectations, appears to increase in the allergic group (Chao-1 richness estimator). This could result from the increase of a heterogeneous set of bacterial taxa for which taxonomic annotation is missing, presumably mostly low-abundance lineages, based on how the Chao-1 index is calculated 58 .
As to the taxonomic identity of these taxa, a systematic survey to evaluate commonly used 16S rRNA "universal" primers on gene sequences from over 6,000 assembled metagenomes has recently shown that > 70% of the bacterial clades systematically under-represented or missed in amplicon-based studies belong to Candidate Phyla Radiation or CPR 59 . CPR is a large monophyletic radiation of ultrasmall bacteria (> 73 phyla) believed to encompass > 25% of taxonomic diversity within the domain, with unique, and still poorly understood, genomic, metabolic and lifestyle features 60,61 . Indeed, a phylogenetic analysis confirmed this hypothesis and showed that sequences unresolved by the V3-V4 metagenomics, and doubled in ALL patients, clusterized within a CPR phylum, namely Candidatus Gracilibacteria (GNO2). This phylum, and others within CPR, as Candidatus Saccharibacteria (TM7), are stable components of the human microbiota, particularly abundant in the oral environment 62 . Interestingly, we have detected (and probably under-estimated) TM7 among the phyla, families, and genera with abundance > 3%, with a trend to increase in patients suffering from food allergies. This trend is in agreement with the available data on TM7, which, although scarce, are virtually the only available on the role of CPR bacteria in the ecology of the human microbiota 63,64 . TM7 bacteria increase in dysbiotic microbiomes and inflammatory environments, both in oral pathologies (e.g., periodontitis, gingivitis) and non-oral conditions (e.g., intestinal autoimmune disorders such as Inflammatory Bowel Disease, IBD), in which an increased TM7 diversity has also been reported 64 . The most immediate conclusion would be labeling TM7 as a pathogenic group. However, this idea has been recently ruled out by Chipashvili and colleagues 63 who used mouse models to show that, instead, TM7 bacteria appear to attenuate oral inflammatory phenomena. This is largely dependent on their peculiar lifestyle as episymbionts of other bacteria, often pathobionts as Actinomyces spp., and their ability to modify traits of their hosts' pathogenicity 63 . Finally, it has been shown that TM7 bacteria themselves present immunomodulatory features (e.g., they suppress Tumor Necrosis Factor-α, TNF-α expression in human macrophages) 64 . In light of these considerations, the fact that Candidatus Saccharibacteria (TM7) and other CPR bacteria, including Candidatus Gracilibacteria (GNO2), are abundant in the oral microbiota of children suffering from food allergy deserves further attention and research on their role in this pathological context.
As well, it would be of interest in future research the measurement of immune parameters in saliva or at a systemic level and their link with taste perception or microbiota composition, whose lack of assessment in the present study has to be mentioned as a limitation.

Conclusions
In conclusion, interest in the sensory field with a specific focus on allergic diseases and food allergy is incipient, and mechanisms underlying taste performance are far from being understood. To date, this is the first study to put emphasis on the link between the sensory system and food allergy, from a one-health approach. Our findings indicate that the food allergic condition appears to impair taste perception, marginally influence the food neophobia attitude, and is linked to compositional shifts in oral microbiota. However, oral microbiota's role in food allergy remains largely unexplored, and some groups of bacteria (i.e., GNO2, TM7 and possibly other lineages within CPR) certainly deserve further attention and research. Finally, given the contribution of taxonomically unresolved taxa to the differential taxonomic picture of food allergy patients vs controls, a more realistic 16S metagenomic picture of this, as well as of probably several other pathological conditions, would certainly require to use sequencing protocols able to include those portions of the bacterial community lost using standard workflows on the Illumina platform, e.g., exploiting more specific primer pairs and/or sequencing whole 16S amplicons.
Appropriate investigation of these factors and their inter-relationships will require collaborative efforts between multidisciplinary teams and will likely open exciting new approaches to therapeutic interventions.

Materials and methods
Participants. The participants were recruited at the Pediatric Clinic of the V. Buzzi Children's Hospital (Milan, Italy). Allergic children were regularly followed at the clinic since their diagnosis of food allergy. The inclusion criteria were: diagnosis of IgE-mediated food allergy (presence of positive skin prick tests and/or specific IgE), confirmed by an oral food challenge, except in case of a clinical history of anaphylaxis, and allergen avoidance diet since at least 6 months, age range 6-14 years old, and Caucasian ethnic group. We excluded patients treated with medications affecting smell or taste and antibiotics within 2 months before the study. A group of healthy sex-and aged-matched controls were recruited as controls from other clinic departments, using the same exclusion criteria. A matching method was used to precisely match cases with non-allergic controls on the basis of sex (same) and age (within 1 year), thus preventing any bias in analyzing the FP count as physiological sensitivity marker, as well as a cognitive bias in taste recognition responses. Using data from a pilot study and previous research 20 , to detect a difference ≥ 1 in taste recognition ability between the two groups, a total sample size of n = 54 subjects ( Taste sensitivity assessment. Taste recognition ability. Taste sensitivity assessment was performed applying the 'Taste Strips' method 65 , using prefabricated filter papers (Taste Strips, Burghart, Wedel, Germany) impregnated with four increasing concentrations of sweet, sour, salty, and bitter taste qualities, plus two tasteless strips. The detailed protocol used is fully described in 19,20 .
Fungiform Papillae (FP) count. The procedure used to assess FP count was fully described in 20 . In brief, the FP was performed by counting the number of these structures inside three 6 mm-diameter circles virtually drawn on the photo of the subject's anterior part of the tongue, using Adobe Photoshop software (Adobe Systems Incorporated, San Jose, California) 66 . The count inside each circle was repeated twice by two independent examiners (blinded to food allergy status) following the Denver Papillae Protocol 67 . Then, the mean of the two counts was calculated.
Food neophobia. The assessment of children's food neophobia was performed through Italian Children Food Neophobia Scale (ICFNS) 52 . Children were asked to express the level of agreement/disagreement for eight statements, using a 5-point facial expression scale ('very false to me' -'very true for me'). The individual Food Neophobia (FN) score was computed as the sum of given ratings, thus, the scores theoretically ranged from 8 to 40, with higher scores reflecting higher FN levels.
Liking questionnaire. A food-liking questionnaire adapted from 19 was completed by children. The questionnaire consisted of 16 different food items characterized by specific taste profiles (i.e., four basic tastes and fattiness) defined in a previous study 68  To determine whether a between-group effect occurs for age and BMI, the independent samples t-test was used. Similarly, a χ 2 test was used to evaluate differences in sex proportions between the two groups. These analyses were carried out to verify the sex-and age match between allergic and controls.
Group-related (allergic vs controls) and sex-related differences in gustatory ability, expressed as single taste scores (i.e., sweet, sour, salty, and bitter scores, each ranging from 0 to 4) and the cumulative one (total taste To assess whether group-related (allergic vs controls) and sex-related differences were associated with Food neophobia, FN scores were submitted to 2-way ANOVA considering Group (allergic vs controls), sex, and their interaction as factors.
Children's food liking was mapped through a principal component analysis (PCA), considering the 16 food items as rows and children's liking scores as columns. The food-liking scores were double-centered before the analysis 69 . Food's taste quality was coded as binary variables (1 = present, 0 = not present) for each of the four basic tastes and fattiness and entered as supplementary variables. Based on PC1 and PC2 loadings, children were clustered into two liking groups. Independent samples t-tests were used to determine whether a between-group effect occurs for age and BMI. Similarly, χ 2 tests were used to evaluate differences in sex and allergic/control subjects' proportions between the two groups. To explore whether Liking clusters influence gustatory functions (i.e., sweet, sour, salty, bitter taste scores, TTS, and FP count) and personality traits (i.e., food neophobia), separated Mann-Whitney U tests and Student's t-test were performed where appropriate. A p-value of 0.05 was considered significant. The SPSS 27.1 (IBM, Armonk, New York) and XLSTAT Sensory package (version 2021.4.1, Addinsoft, Boston, MA, USA) were used for the data analysis.
The bioinformatics analysis of microbiota sequencing data was based on the Mothur pipeline 70 . Raw FASTQ files were quality-filtered using Trimmomatic 71 and high-quality reads were analysed following the SOP Mothur procedure, as described in 20 . The main ecological indexes of within-sample, α-diversity (Shannon, Chao, inverse Simpson) were computed using Mothur. Diversity in composition among samples (β-diversity) was evaluated by plotting the relative heatmap using the function heatmap.2 of the Gplots R library 72 and the relative Principal Component Analysis (PCoA) using the R library Ade4 73 . The Permanova analysis was performed by using the R library Vegan 74 . Microbial profiles of patients and controls were finally compared to evidence statistically significant differences in bacterial composition and taxa abundances, using Mann-Whitney U test with a significance threshold (p-value) set to 0.05.
After metagenomic analysis, the representative sequence of the most abundant OTUs "unclassified" by the Mothur pipeline, and counted at least 20 times in at least one sample, were selected. These sequences were Blastnsearched against the HOMD 16S database (https:// www. homd. org/) and, for each, the 50 more similar sequences were retrieved. The obtained dataset, including the selected OTUs and HOMD entries, was aligned using Muscle 75 and subjected to Maxumum Likelihood (ML) phylogenetic analysis using RAxML8 76 with 100 pseudobootstraps.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.